Stigmasterol: Remodeling gut microbiota and suppressing tumor growth through Treg and CD8+ T cells in hepatocellular carcinoma

Hepatocellular carcinoma (HCC), the most primary malignant liver tumor and is ranked as the fifth most common malignancy worldwide. Despite various therapeutic approaches being used in clinical practice, the overall effectiveness remains insufficient. Stigmasterol, a compound known for its anti-tumor properties and ability to induce apoptosis in tumor cells, has been found to influenced the composition of the intestinal microbiota. However, the mechanism through which stigmasterol influences the intestinal microbial-host crosstalk in HCC remains elusive. This study was to investigate whether stigmasterol can remodel gut microbiota, and suppress tumor volume by regulating Treg and IFN-γ+ CD8+ cell in the host with HCC. Stigmasterol (at dosages of 0, 50, 100, or 200 mg/kg) was orally administered to Balb/c mice with subcutaneous tumor once every 2 days for 3 weeks. We first found that tumors volume in the group treated with 100 mg/kg stigmasterol were significantly decreased compared with those in the control group (P<0.05), which exhibited a similar effect as the sorafenib treatment in mice with HCC. This resulted in a significant upregulation of Caspase3, Bax, and P53 expressions, as well as a decrease in Cyclin D1 expression, ultimately leading to a reduction in tumor volume. Additionally, stigmasterol can alter the α and β diversity of the intestinal flora and significantly increase the abundance of Lactobacillus_johnsonii, Lactobacillus_murinus, and Lactobacillus_reuteri (P<0.05), which can lead to a decrease in the ratio of regulatory T cells (Tregs) to CD8+ T cells in the intestinal tract and tumor tissue, and consequently enhance immune response in the tumor microenvironment (TME) in the host with HCC. In this study, we initially utilized different dosages of stigmasterol to intervene in mice with HCC and confirmed its inhibitory effects on tumor growth in vivo, and discovered that stigmasterol affected Lactobacillus johnsonii, Lactobacillus murinus, and Lactobacillus reuteri, resulting in an increased proportion of IFN-γ+ CD8+ T cells and Treg cells in both the intestinal mucosa and tumor tissues, and ultimately leading to increased levels of apoptotic proteins and the subsequent death of tumor cells, which shed light on the effect of stigmasterol on host intestinal tissue and intratumoral immune cells by reshaping the intestinal microbiota, and provide a theoretical foundation for the potential clinical application of stigmasterol in the treatment of HCC.